As an indicator for showing a biological state, the glycation degrees of various kinds of proteins are assayed. Among them, a glycation degree of hemoglobin (Hb) in a blood cell, in particular, HbA1c is used as an important indicator in diagnoses, treatments and the like for diabetes, because the HbA1c reflects histories of an in-vivo blood glucose level. The HbA1c has a structure in which a glucose binds to a β-chain N-terminal amino acid (valine) of HbA (α2β2), and its glycation degree (%) is represented by a ratio (%) of a HbA1c amount with respect to a total Hb amount.
In general, HbA1c is assayed by a high performance liquid chromatography (HPLC), an immunization method, an enzymatic method, an electrophoresis or the like. For example, a method, which includes cleaving a β-chain N-terminal Hexpep of Hb and a glycated Hexpep as its glycated product by a protease Glu-C, separating and refining by the HPLC, and subsequently determining a quantity thereof by capillary electrophoresis or LC-MS, is disclosed as a reference method of HbA1c (Non-patent Document 1). Among them, the enzymatic method is, for example, an assay method as described below. Firstly, a fructosyl amino acid oxidase (hereinafter, referred to as a “FAOD”) is allowed to act on a glycated part of Hb, thereby generating hydrogen peroxide. An amount of this hydrogen peroxide corresponds to an amount of the glycated Hb. Then, a peroxidase (hereinafter, referred to as a “POD”) and a chromogenic substrate that develops color by oxidation are added further to this reaction liquid, so that a redox reaction occurs between the hydrogen peroxide and the chromogenic substrate with the POD as a catalyst. Then, a glycation amount is obtained by assaying a chromogenic level of the chromogenic substrate, and as a result, a HbA1c value (%) can be calculated from the glycation amount and the total Hb amount.
As described above, since the HbA1c is characterized in the glycation of the β-chain N-terminal valine, the FAOD is desired to act on the N-terminal glycated valine efficiently. However, since the FAOD is not likely to act directly on a protein, a method of cleaving the β-chain N-terminal of the Hb by protease treatment and allowing the FAOD to act on the β-chain N-terminal directly is attempted generally. More specifically, methods of using an endo-type or exo-type protease (Patent Document 1), serine carboxypeptidase (Patent Document 2), a protease that liberates amino acid whose α-amino group is glycated (Patent Documents 3 to 5), a protease that cleaves glycated amino acid or a glycated peptide at a β-chain N-terminal by a stronger action than that at an α-chain N-terminal (Patent Document 6) and the like are reported (Patent Documents 7 to 10). Moreover, a method of denaturating hemoglobin by boiling with urea added, and treating with a protease also is reported.
However, according to these methods, a long period of time is required for the protease treatment, so that it is difficult to carry out the examination swiftly. It is reported that, also in the above-described capillary electrophoresis and the protease treatment for the LC-MS, the treatment at 37° C. for 18 hours is required. Moreover, an example of carrying out the treatment with molsin under an acid condition at pH of 3 for about 1 hour is disclosed (Patent Document 11) as an example of reducing the time of the protease treatment, but substantial neutrality is preferable for effecting the enzyme reaction, and the treatment under such a condition is not disclosed. Also in the case of assaying a glycation degree of a part of the Hb other than the β-chain N-terminal, there is a problem in the speed of the cleavage, similarly to the case of the HbA1c described above.
Non-patent Document 1: Masao UMEMOTO, “IFCC Method of HbA1c for International Standardization”, Clinical examination, Vol. 46, No. 7, July 2002
Patent Document 1:WO 1997/013872 A1Patent Document 2:JP 2001-57897 APatent Document 3:WO 2000/50579 A1Patent Document 4:WO 2000/61732 A1Patent Document 5:JP 2002-315600 APatent Document 6:JP 2004-344052 APatent Document 7:JP 5(1993)-192193 APatent Document 8:JP 10(1998)-33177 APatent Document 9:JP 10(1998)-33180 APatent Document 10:JP 2004-333452 APatent Document 11:JP 2001-95598 A